Compounds and methods for treating or preventing disease conditions associated with alpha-1-antitrypsin

ABSTRACT

Pharmacological chaperone compounds and methods for the treatment of an individual having, or at risk of having, a disease condition associated with alpha-1-antitrypsin by using said compounds are disclosed. In particular, such methods are useful for the treatment or prevention of lung disorders associated with alpha-1-antitrypsin deficiency as well as liver disorders associated with an excess of alpha-1-antitrypsin. Suitable pharmacological chaperones include peptides and low-molecular weight compounds. Also provided is an assay for determining whether a test compound modulates alpha-1-antitrypsin activity.

FIELD OF THE INVENTION

The present invention relates to compounds and methods of using the samefor treating and/or preventing disease conditions associated withα-1-antitrypsin.

BACKGROUND

Many human genetic disorders are caused by mutations that impair proteinfolding and trafficking. Even though the mutated proteins may beproduced in normal amounts and may even be functionally competent,problems can arise because the mutated proteins do not fold properlyand/or are not processed and trafficked correctly. Consequently, suchproteins do not reach their intended cellular location and tend toaccumulate in the endoplasmic reticulum (ER) or other organdies wherethey are prone to aggregation. The relative importance of thesecontributions to cellular dysfunction and disease varies among diseases,and may even differ from patient to patient and potentially from celltype to cell type. There are some conditions where loss of proteinfunction is the primary cause of disease, and others for which atoxic-gain-of function is caused by aggregation, and excessive ERretention is the primary source of pathology. In the case ofα-1-antitrypsin deficiency, both loss-of-function andtoxic-gain-of-function contribute to disease pathology.

Alpha-1-antitrypsin is a protein made in hepatocytes and secreted fromthe liver into the blood where it functions to limit neutrophil elastaseactivity in the lung. A deficiency in α-1-antitrypsin can lead toemphysema, as a result of increased degradation of lung connectivetissue. In many patients, alpha-1-antitrypsin deficiency is caused by aE342K missense mutation therein, referred to as the Z mutant (Z-AT). Thequality control mechanisms of the ER lead to retention and accumulationof Z-AT in hepatocytes, causing damage to the liver and reducing plasmalevels of alpha-1-antitrypsin as a result of reduced secretion into theblood. Thus, lung disease associated with alpha-1-antitrypsin deficiencyis caused by a loss-of-alpha-1-antitrypsin function, while liver diseaseoccurs when the Z-AT accumulates to toxic levels in liver cells(toxic-gain-of-function). Since monomeric Z-AT retains the same specificactivity as wild type α-1-antitrypsin (M-AT), treatment strategies thatincrease secretion of Z-AT by reducing its ER retention should protectagainst both liver and lung damage.

Studies have demonstrated that some compounds, such as 4-phenylbutyricacid, can increase secretion of Z-AT from cells. Additionally, somesmall peptides (e.g., Ac-TTAI-NH₂) and citrate have been shown to blockin vitro polymerization of Z-AT. However, the use of such osmolytes topromote protein folding is very limited as they require very highcellular concentrations and lack target specificity. Desirably, toeffect protein folding and secretion in vivo, a compound must be able topenetrate the ER of liver cells, have a high affinity for Z-AT, andblock polymerization thereof with minimal toxicity and side effects.

Current therapy for conditions associated with α-1-antitrypsindeficiency is limited to protein replacement therapy with M-AT derivedfrom human plasma, typically dosed on a weekly basis. While such therapyis effective for lung pathology including emphysema, it has no effect onliver disease caused by the accumulation of polymerized Z-AT in the ERof hepatocytes. For the 10-15% of homozygotes for Z-AT afflicted withearly-onset liver disease including cirrhosis and hepatocellularcarcinoma, liver transplantation is the only treatment option available.Furthermore, accumulation of polymerized Z-AT in lung epithelium has achemoattractant effect on neutrophils, which may cause furtherdestruction of connective lung tissue. Thus, there is a need fortherapeutics and methods that address disease conditions associated withboth alpha-1-antitrypsin deficiency as well as toxic accumulation ofalpha-1-antitrypsin.

SUMMARY

The present invention provides compounds and methods for using thesecompounds to treat an individual having or at risk of having a diseasecondition associated with alpha-1-antitrypsin comprising administeringto the individual an effective amount of a pharmacological chaperone. Inparticular, such methods are useful for the treatment and/or preventionof lung disorders associated with alpha-1-antitrypsin deficiency. Suchmethods are also useful for the treatment and/or prevention of liverdisorders associated with an excess of alpha-1-antitrypsin. The presentinvention also provides an assay for determining whether a test compoundmodulates alpha-1-antitrypsin activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a dose response curve from an alpha-1-antitrypsin elastaseinhibition assay.

FIG. 2 shows inhibition of elastase activity by alpha-1-antitrypsin inthe presence of peptides that inhibit polymerization.

FIG. 3 is a Western blot with Z-AT exposed to one of several acetylatedpeptides or control (no peptide), wherein the degree ofalpha-1-antitrypsin polymerization is evident.

FIG. 4 is a graph illustrating the level of human alpha-1-antitrypsindetected by ELISA in media from cultured cells expressing the Z mutantform, S mutant form or wild type (M) form of human alpha-1-antitrypsin.

FIG. 5 is a graph of elastase activity by measuring the relativefluorescence versus concentration of inhibitor.

FIG. 6 shows a Western Blot of lysates and media from SF-CHO and HeLainducible Z-AT expressing cell lines.

FIG. 7 is a computational simulation of Ac-Thr-Glu-Val-Ala-NH2,illustrating 11 hydrogen bonds between s5a and s3a of beta-sheet A.

FIG. 8 shows a Western Blot of wild type, Z mutant and the doublecysteine Z mutant of alpha-1-antitrypsin polymerization.

FIG. 9 shows a Western Blot of the intracellular levels of ZAT in ZATinducible CHO cells after treatment with compound V and VI at 100 μM.

FIG. 10 shows a Western Blot of the intracellular levels of ZAT in ZATinducible CHO cells after treatment with compounds V and VI at 1 mM.

FIG. 11 shows extracellular levels of alpha-1-antitrypsin activity inmedia for ZAT inducible cell lines treated with compound V.

FIG. 12 shows extracellular activity of ZAT after 1 mM treatment withcompounds V and VI.

FIG. 13 shows a Western Blot of intracellular levels of ZAT after 1 mMtreatment with compounds V and VI.

FIG. 14 is a graph of normalized densitometry, showing intracellularlevels of ZAT after 1 mM treatment with compounds V and VI.

FIG. 15 shows an in vitro polymerization assay, showing polymerizationof ZAT after treatment with compounds I-VI and Ac-TTAI-NH2.

FIG. 16 is a computational simulation of compound I forming hydrogenbonds between strands s3a and s5a.

DETAILED DESCRIPTION

The present invention provides compounds, known as pharmacologicalchaperones, and methods for using these compounds to prevent and/ortreat disease conditions associated with alpha-1-antitrypsin.Pharmacological chaperones include peptides and small molecules, whichselectively bind to a target protein and increase protein stabilityand/or proper trafficking thereof such that the target protein can passthe ER quality control system and function at its proper site. Thus, theadministration of a pharmacological chaperone can increase proteinlevels and cellular activity of the target protein. Additionally,administration of a pharmacological chaperone reduces ER accumulation ofthe target protein as well as aggregation thereof and associated stresson cells.

Though not meant to be limited by any theory with the subject invention,the compounds of the present invention are believed to bind to a site onalpha-1-antitrypsin, (specifically, the A beta-sheet 4, morespecifically P8-4 of the reactive center loop) and thereby abolishpolymerization of alpha-1-antitrypin. Furthermore, non-acetylated formsof such compounds are believed to dissociate from alpha-1-antitrypsin.

DEFINITIONS

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification, unless otherwise limited in specificinstances, either individually or as part of a larger group.

As used herein, “α-1-antitrypsin,” or “alpha-1-antitrypsin” or “AAT”refers to a protease inhibitor which inhibits elastase, among otherproteases.

As used herein, “disease condition associated with α-1-antitrypsin”refers to an autosomal genetic disorder leading to an accumulation ofα-1-antitrypsin and/or deficiency of α-1-antitrypsin in one or moreorgans such that toxic levels of α-1-antitrypsin activity and/or adeficiency of α-1-antitrypsin activity results. Diseases associated withα-1-antitrypsin include, but are not limited to, cirrhosis, chronicobstructive pulmonary disease, pneumothorax, asthma, Wegener'sgranulomatosis, pancreatitis, gallstones, bronchiectasis, pelvic organprolapse, primary sclerosing cholangitis, autoimmune hepatitis,emphysema (predominantly involving the lower lobes and causing bullae),cancer (including hepatocellular carcinoma (liver), bladder carcinoma,gallbladder cancer, lymphoma and lung cancer).

As used herein, “Z mutation” refers to the E342K (Glu342Lys) missensemutation, which causes alpha-1-antitrypsin deficiency. The mutated formof alpha-1-antitrypsin can be abbreviated as “ZAT” or “Z-AT.”

As used herein, “MAT” refers to wild-type alph-1-antitrypsin protein,produced by the so-called normal allele.

As used herein, “treating” means to ameliorate one or more symptomsassociated with the referenced disorder.

As used herein, “preventing” means to mitigate a symptom of thereferenced disorder.

As used herein, “an effective amount” means an amount effective toprevent and/or treat a patient at risk for developing or diagnosed withthe referenced disorder, and thus producing the desired therapeuticeffect.

Furthermore, where “R” is used, it is understood by a person of ordinaryskill in the art that R, R₂, R₃, etc. will not be selected such that anunstable molecule will result.

α-1-Antitrypsin Activity Assay

An elastase inhibition assay was developed to measure the effects of atest compound on the inhibitory activity of secreted Z-AT. In oneembodiment, to detect elastase activity, bovine elastin was labeled withBODIPY, which causes the fluorescence of the conjugate to be quenched.This non-fluorescent substrate was digested by elastase to yield highlyfluorescent fragments. Alternatively, the non-fluorescent substrate maybe digested by another protease to yield fluorescent fragments. Theactivity of α-1-antitrypsin was monitored by measuring a decrease influorescent signal, indicative of the elastase inhibitory activity ofα-1-antitrypsin. A test compound was added to determine its effect onthe elastase inhibitory activity of α-1-antitrypsin. In one embodiment,the resulting fluorescence was monitored in a Perkin Elmer Victor Vplate reader using absorption and emission filters for fluorescein(435/535 nm).

Cell Based Assays Serum Free CHO Cell Lines.

Serum free CHO cell lines were developed, expressing M-AT, Z-AT(Glu342Lys) and the S mutation (Glu264Val), so that secretedalpha-1-antitrypsin activity could be measured directly from mediawithout interference from bovine, alpha-1-antitrypsin introduced intothe media from serum. Briefly, cDNA was isolated by PCR from firststrand human liver cDNA using conventional cloning methods. The Z-AT andS-AT mutations were generated using Quick Change mutagenesis kitaccording to the manufactures (Stratagene) instructions. Plasmidsexpressing M-AT, Z-AT or S-AT under control of the CMV promoter weretransfected into CHO cells that were adapted to serum free conditions.This cell-based model makes it possible to ascertain the ability of atest compound to effect alpha-1-antitrypsin activity by ascertaining theimpact on elastase inhibitory activity of alpha-1-antitrypsin in mediafrom cultured cells. ELISA and activity levels of alpha-1-antitrypsinboth indicate that Z-AT is secreted into the media at concentrations10-fold below M-AT corresponding to the decrease in levels observed inPiZZ homozygote patients.

The media and lysates from the Z-AT and M-AT cell lines were analyzed ona native gel followed by western blotting to determine the extent ofpolymerization. Z-AT that is secreted into the media from the serum freeCHO line is essentially all polymerized. While lysates from Z-AT andM-AT cell lines do contain a small percentage of monomericalpha-1-antitrypsin, the majority of material is polymerized. Incontrast, monomeric M-AT was detected from wild type cell line asexpected.

In addition, inducible CHO and HeLa cell lines expressing Z-AT and M-AThave been generated for screening purposes. While the inducible CHO cellline secretes Z-AT, the HeLa inducible cell line retains essentially allthe Z-AT intracellularly, as shown in FIG. 6.

Analysis of Intracellular and Extracellular Z-AT in Constitutive andInducible Cell Lines

Additional cell lines to screen for pharmacologic chaperones and toidentify surrogate markers of Z-AT expression were developed using aninducible promoter system. The inducible systems are useful fordetermining if particular chaperones need to be present prior to Z-ATtranslation in the endoplasmic reticulum to induce secretion of Z-ATand/or inhibit The detection limit of this embodiment of the assay forα-1-antitrypsin is approximately 16 ng/ml and the fluorescence wasrelatively insensitive to pH between a pH of about 3 to a pH of about 9.FIG. 1 displays a typical dose response curve using increasingconcentrations of M-AT and an elastase concentration of about 2.5 mU/ml.This level of sensitivity for detecting the activity of α-1-antitrypsinis particularly important when screening numerous test compounds, sincerelatively small cell numbers are typically used for cell basedscreening assays. FIG. 5 also uses this assay to compare activity ofcells transiently transfected with α-1-antitrypsin M, S and Z alleles.

Human Alpha-1-Antitrypsin Specific ELISA.

An ELISA that is commercially available (Alpco) was tested forsensitivity and specificity to human M-AT to test for stimulation ofZ-AT secretion from cells and plasma in vivo. Testing confirmed thatthis ELISA assay is highly specific for human alpha-1-antitrypsin andhas no cross reactivity with mouse serum. The level of detection forthis assay is 3-4 ng/ml. The sensitivity of this assay along with itsspecificity makes it a useful quantitative assay for cell based and invivo testing of plasma. FIG. 4 shows the results of an ELISA analysisdetecting the presence of human alpha-1-antitrypsin in media fromcultured cells expressing the Z mutant form, S mutant form or wild type(M) form of alpha-1-antitrypsin.

In Vitro Polymerization Assay.

An in vitro polymerization assay was employed to screen the ability of apharmacological chaperone to inhibit polymerization. Notably, theZ-mutation, E342K, lowers the kinetic barrier to polymerization relativeto M-AT, causing the characteristic polymerization of Z-AT. In brief,M-AT or Z-AT was incubated at 37° C. for 4 hours, followed by 30 minutesat 62° C. The samples were then run on a Bis-Tris native gel and eitherblotted onto PVDF or detected with colloidal blue staining. Peptides at1 mM were incubated with equal amounts (50 ng) of Z-AT for 4 hours at37° C., followed by a 62° C. pulse for 30 minutes. Samples wereseparated on a 4-12% Bis-Tris native gel, blotted onto PVDF and the blotprobed with a human specific antibody for alpha-1-antitrypsin.Additionally, in the case of samples from human plasma, a western blotwas performed to assess the level of alpha-1-antitrypsin.polymerization. The inducible HeLa cell line retains Z-ATintracellularly as little Z-AT can be detected in the media. The Z-ATcontained in lysates is apparently not processed to complex N-glycans asthe molecular weight is slightly lower than M-AT purified from plasma,which is consistent with the findings of others. The intracellularmaterial is endo H sensitive indicative of high mannose N-linkedglycans.

Serum Free CHO and HeLa cells in which expression of Z-AT is undercontrol of the tetracycline repressor were grown to confluence. Toanalyze the intra- and extracellular production of Z-AT, media wascollected and cells were lysed in non-ionic detergent. Media and lysateswere analyzed on a NuPAGE SDS gel followed by western blotting utilizinga human specific sheep anti-human alpha-1-antitrypsin polyclonalantibody.

The lack of N-linked processing in intracellular Z-AT is also clear inthe serum free CHO cell line. These data demonstrate that these celllines retain Z-AT intracellularly, most likely in the endoplasmicreticulum. Furthermore, the Z-AT that is retained or secreted is in apolymeric state based on native gels followed by western blotting. Thesecell lines represent useful screening tools for the identification ofsmall molecules that inhibit polymerization and stimulate secretion ofactive human Z-AT.

Introduction of Disulfide Bond Between Strands s5a and s6a of Beta-SheetA Inhibits Polymerizations of ZAT in Mammalian Cell Lines and IncreasesSecretion of Active ZAT

There is a variant of the Z mutation which features a double cysteinemutation. In addition to the E342K mutation, there are also T339C andS292C mutations. This double cysteine mutation also showsalpha-1-antitrypsin polymerization. However, it shows lesspolymerization as compared to the normal Z mutation. FIG. 8 shows awestern blot of the three forms. There is some polymerization in thedouble cysteine mutation, less than the Z mutant, but more than the wildtype. Thus, introduction of a disulfide bond between strands s5a and s6adecreases polymerization of ZAT, and increases secretion of active ZATin the media of mammalian cells (CHO and HeLa).

Peptides that Inhibit Polymerization of Z-AT

Peptides that inhibit the polymerization of Z-AT includeAc-Thr-Glu-Ala-Ala-Gly-NH2, Ac-Thr-Ser-Ala-Ala-NH2,Ac-Thr-Glu-Val-Ala-NH2 and Ac-Thr-Glu-Ala-Ala-NH2. Such peptides can besynthesized using conventional methods known to one of skill in the art.One embodiment of peptide synthesis uses standard Fmoc chemistry withpeptide amide linker (PAL) resin as solid support on a peptide synthesisapparatus. After deprotection of the N-Fmoc group on a solid supportwith 20% (v/v) piperidine in dimethylformamide (DMF), the resin isplaced in a reaction vessel. The selected amino acids are coupled to theresin in the vessel. All coupling reactions are performed in DMF atambient temperature for 2 hrs using a 4-fold excess of Fmoc-protectedamino acids relative to resin loading. Each coupling step is monitoredby a ninhydrin test to ensure that the amino acids are completelyincorporated into the forming peptides. After completion of coupling,the resin from the vessel is thoroughly washed, mixed, deprotected andreplaced into the reaction vessel. For the acetylation on N terminus,the resin is soaked in acetic anhydride/diisopropylethylamine/DMF for 2hrs. Final release of the peptides from the resin and side chaindeprotection is concomitantly achieved with 95% (v/v) trifluoroaceticacid in water for 2 hrs. All peptides are then lyophilized three timesprior to use.

FIG. 3 shows a western blot of ZAT in the presence of several acetylatedpeptides as well as control (no peptide). Ac-TSAA-NH2, Ac-TEVA-NH2,Ac-TEAA-NH2 and Ac-TEAAG-NH2 show significantly less alpha-1-antitrypsinpolymerization as compared to the other peptide compounds whereinalpha-1-antitrypsin polymerization was similar to untreated Z-AT. Thereduction of alpha-1-antitrypsin polymerization demonstrates the abilityof peptides depicted in Table 1 to be used as a treatment to reduceaccumulation of Z-AT in the liver.

TABLE 1 Acetylated Peptides that Inhibit Polymerization

Ac-Thr-Ser-Ala-Ala-NH2 (Ac-TSAA-NH2)

Ac-Thr-Glu-Val-Ala-NH2 (Ac-TEVA-NH2)

Ac-Thr-Glu-Ala-Ala-NH2 (Ac-TEAA-NH2)

Ac-Thr-Glu-Ala-Ala-Gly-NH2 (Ac-TEAAG-NH2)

FIG. 2 shows the inhibition of elastase activity by alpha-1-antitrypsinin the presence of peptides that inhibit polymerization. The AAT assaydisclosed above was used. This graph reveals that alpha-1-antitrypsin inthe presence of Ac-Thr-Glu-Val-Ala-NH2 inhibits elastase activitysignificantly, in fact to a level comparable to that of wild typealpha-1-antitrypsin. FIG. 7 shows a computational simulation ofAc-Thr-Glu-Val-Ala-NH2 bound to the β-sheet using BioPredict. Thesimulation suggests Ac-Thr-Glu-Val-Ala-NH2 forms eleven hydrogen bondsbetween the s5a and s3a strands of Z-AT. Therefore,Ac-Thr-Glu-Val-Ala-NH2 inhibits polymerization, but does not inhibitalpha-1-antitrypsin activity at concentrations required to inhibitpolymerization. Thus, Ac-Thr-Glu-Val-Ala-NH2 could help both with aidingthe removal of accumulated ZAT from the ER and allowing the ZAT tofulfill its protease inhibition function. This makesAc-Thr-Glu-Val-Ala-NH2 a potentially good agent to treat both liver andlung-related diseases caused by alpha-1-antitrypsin.

To increase membrane permeation and plasma stability, peptides may becyclized. One embodiment of such a cyclization is to add a lactonegroup. An example of a prophetic synthetic scheme for cyclization ofAc-Thr-Thr-Ala-Thr-NH2 by creating a macrolactone is as followsaccording to Scheme 1:

Additional Compounds

Additionally, several low molecular weight compounds (Compounds I-VI)were discovered which increase hydrogen bonding between s3a and s5a ofbeta-sheet A. FIG. 16 shows a computational simulation of compound I.Compound I forms seven potential hydrogen bonds between strands s3a ands5a. Furthermore, two compounds, compounds V and VI, decreaseintracellular levels of ZAT. These compounds are shown in Table 2, alongwith structural data for each compound. FIG. 9 shows a western blot ofthe intracellular levels of ZAT after treatment with 100 μM of compoundsV and VI. Compounds V and VI show lower levels of ZAT. Similarly, FIG.10 shows the intracellular levels of ZAT after treatment with compoundsV and VI at a concentration of 1 mM. The levels of intracellular levelsof ZAT are even lower when treated at the higher concentration. FIG. 11is a graphical representation of extracellular levels of ZAT andalpha-1-antitrypsin activity in ZAT inducible CHO cells after treatmentwith compound V. FIG. 11 shows higher activity levels with highertreatment with compound V. FIG. 12 is a similar graph, showingextracellular activity of ZAT after 1 mM treatment with compounds V andVI. FIG. 13 depicts a western blot of intracellular levels of ZAT after1 mM treatment. Compounds V and VI show significant reduction in ZATlevels intracellularly. FIG. 14 is a graph of showing intracellularlevels of ZAT after 1 mM treatment with compounds V and VI. Levels arelower after treatment with compounds V and VI. FIG. 15 is an in vitropolymerization assay with compounds I-VI, as well as Ac-TTAI-NH2, avehicle control and non-treated alpha-1-antitrypsin. There is anincrease in polymerization after treatment with the low-molecular weightcompounds.

TABLE 2 Small Molecules That Decrease Intracellular Levels of ZAT LC-MSand ¹H-NMR Structure Data

Compound I C₁₂H₁₆N₄0₅, MW = 296; LC-MS: M + H = 297, M + Na = 319;¹H-NMR (600 MHz, DMSO) 9.78 (s, 1H), 8.08 (s, 1H), 7.45 (d, 2H), 7.32(t, 2H), 7.27 (t, 1H), 7.21 (s, 1H), 7.12 (s, 1H), 6.30 (d, 1H), 6.15(d, 1H), 5.01 (d, 1H), 4.91 (t, 1H), 4.04 (m, 1H), 3.60 (m, 1H), 3.47(m, 1H).

Compound II C₁₂H₁₆N₄0₅, MW = 296; LC-MS: M + H = 297, M + Na = 319;¹H-NMR (600 MHz, DMSO) 9.78 (s, 1H), 8.08 (s, 1H), 7.45 (d, 2H), 7.32(t, 2H), 7.27 (t, 1H), 7.21 (s, 1H), 7.12 (s, 1H), 6.30 (s, 1H), 6.15(d, 1H), 5.01 (d, 1H), 4.91 (t, 1H), 4.04 (m, 1H), 3.60 (m, 1H), 3.47(m, 1H)

Compound III C₁₃H₁₈N₄0₅, MW = 310; LC-MS: M + H = 311, M + Na = 333;¹H-NMR (600 MHz, DMSO) 9.79 (s, 1H), 8.12 (s, 1H), 7.45 (d, 2H), 7.32(t, 2H), 7.27 (t, 1H), 7.09 (d, 2H), 6.18 (s, 2H), 5.01 (d, 1H), 4.92(d, 1H), 4.03 (m, 1H), 3.92 (m, 1H), 0.98 (d, 3H).

Compound IV C₁₃H₁₈N₄0₅, MW = 310; LC-MS: M + H = 311, M + Na = 333;¹H-NMR (600 MHz, DMSO) 9.80 (s, 1H), 8.12 (s, 1H), 7.45 (d, 2H), 7.32(t, 2H), 7.27 (t, 1H), 7.09 (d, 2H), 6.17 (s, 2H), 5.01 (d, 1H), 4.92(d, 1H), 4.03 (t, 1H), 3.93 (m, 1H), 1.00 (d, 3H).

Compound V C₁₃H₁₈N₄0₇, MW = 342; LC-MS: M + H = 343, M + Na = 365;¹H-NMR (600 MHz, DMSO) 9.66 (s, 1H), 8.89 (s, 1H), 8.05 (s, 1H), 7.22(d, 1H), 7.12 (s, 1H), 6.87 (s, 1H), 6.82 (t, 2H), 6.30 (s, 1H), 5.95(s, 1H), 4.92 (s, 1H), 4.86 (d, 1H), 4.04 (m, 1H), 3.74 (s, 3H), 3.60(t, 1H), 3.49 (m, 1H).

Compound VI C₁₄H₂₀N₄0₇, MW = 356; LC-MS: M + H = 357, M + Na = 379;¹H-NMR (600 MHz, DMSO) 9.66 (s, 1H), 8.89 (s, 1H), 8.09 (s, 1H), 7.10(s, 1H), 7.08 (s, 1H), 6.87 (s, 1H), 6.82 (m, 2H), 6.19 (s, 1H), 5.96(s, 1H), 4.91 (m, 1H), 4.86 (d, 1H), 4.02 (m, 1H). 3.94 (m, 1H), 3.73(s, 3H), 1.01 (m, 3H).

Synthesis

Scheme 2 shows one process specifically for the synthesis of compoundsIII and IV. However, it is easily modified for other low molecularweight compounds, including compounds I, II, V and VI by starting withthe corresponding starting material noted in Table 3 below. To asolution of methanol (50 ml), SOCl₂ (10 ml) was added dropwise at 0° C.,the solution was allowed to stir for 1 hr at room temperature, thenthreonine (5.1 g, 42.9 mmol) was added, the mixture was stirred for 7hrs at 38° C. and concentrated to dryness, the crude product waspurified by re-crystallization form petroleum ether (5 ml) and ether (1ml) to afford pure product 12-M-1 (5.0 g; yield: 88%).

To a solution of 12-M-1 (7.3 g, 42.9 mmol) and K₂CO₃ (14.8 g, 107 mmol)in THF (100 ml), (Boc)₂O (11.2 g, 51.5 mmol) was added dropwise at 0°C., the solution was allowed to stir for 3 hrs at room temperature, thenthe solution was concentrated to dryness to get the crude product, thecrude product was purified by silica gel chromatography eluted with(EtOAc: PE=1:4) to give product 12-M-2 (9.9 g; yield: 99%) as oil.

The solution of 12-M-2 (233 mg, 1.0 mmol) in methanol amine solution (10ml), the solution was allowed to stir over night at 0° C., then thesolution was concentrated to dryness to get the crude product 12-M-3,the crude product was used directly for the next step withoutpurification.

To a solution of 12-M-3 and pyridine (119 mg, 1.5 mmol) in THF (10 ml),Fmoc-Cl (280 mg, 1.1 mmol) was added dropwise at 0° C., the solution wasallowed to stir for 4 hrs at room temperature, then the solution wasconcentrated to dryness to get the crude product, the crude product waspurified by silica gel chromatography eluted with (EtOAc: PE=1:10) togive product 12-M-4 (394 mg; yield: 89.6%) as white solid.

To a solution of 12-M-4 (390 mg, 0.80 mmol) in DCM (5 ml), TFA (2.5 ml)was added dropwise, the solution was stirred for 4 hrs at roomtemperature, then the solution was concentrated to dryness to get thecrude product 12-M-5, the crude product was used directly for the nextstep without purification.

To a solution of 12-M-5 in DCM (15 ml), saturated solution of sodiumbicarbonate (6 ml) was added, the solution was stirred for 20 mins. Thentriphosgene (89 mg, 0.3 mmol) was added at 0° C. and the solution wasstirred for 20 mins at 0° C. The mixture was extracted with DCM (20 ml),the organic layers was dried and concentrated to dryness to get thecrude product 12-M-6, the crude product was used directly for the nextstep without purification.

To a solution of (S)-methyl 2-hydroxy-2-phenylacetate (2.4 g, 14.5 mmol)in methanol (15 ml), hydrazine hydrate (4.6 g, 150 mmol) was addeddropwise, the solution was allowed to stir for 4 hrs at roomtemperature, then the solution was concentrated and extracted with DCM,the organic phase was dried over Na₂SO₄ and evaporated to dryness to getthe crude product, the crude product was purified by silica gelchromatography eluted with (DCM: MeOH=20:1) to give product 12-M-7 (1.0g; yield: 42%) as white solid.

A solution of 12-M-6 and 12-M-7 (100 mg, 0.6 mmol) in DCM (25 ml) wasrefluxed for 2 hrs, then the solution was stirred over night at roomtemperature. The solution was concentrated to dryness to get the crudeproduct, the crude product was purified by silica gel chromatographyeluted with (DCM: MeOH=10:1) to give product 12-M-8 (150 mg; yield: 47%)as white solid. LC-MS: [M+H]⁺=533, [M+Na]⁺=555 (C₂₈H₂₈N₄O₇ MW=532).

A solution of 12-M-8 (60 mg) and piperidine (0.5 ml) in DCM (20 ml) wasstirred for 10 mins at room temperature. The solution was concentratedto dryness to get the crude product, the crude product was purified bysilica gel chromatography eluted with (DCM: MeOH=10:1) to give compoundIII and IV.

TABLE 3 Compounds for Synthesis Name Structure Benzyloxycarbonylchloride

L-Alaminamide hydrochloride

L-Serinamide hydrochloride

L-Threoninamide hydrochloride

R-(−)-Mandelic acid

S-(+)-Mandelic acid

4-methoxy-3- hydroxymandelic acid

What is claimed is:
 1. A method of treating an individual having or atrisk of having a disease condition associated with α-1-antitrypsincomprising administering to the individual an effective amount of apharmacological chaperone.
 2. The method of claim 1, wherein thepharmacological chaperone is:

wherein: R1 is —(CH₂)₂—COOH or —CH₂—OH R2 is —CH₃ or —CH—(CH₃)₂,

wherein: R₃ is —H or —CH₃ R₄ is —H or —OCH₃ R₅ is —H or —OH, orcombinations of two or more thereof.
 3. The method of claim 1, whereinthe pharmacological chaperone is: Ac-Thr-Glu-Ala-Ala-NH2,Ac-Thr-Glu-Ala-Ala-Gly-NH2, Ac-Thr-Ser-Ala-Ala-NH2,Ac-Thr-Glu-Val-Ala-NH2 or combinations of two or more thereof.
 4. Themethod of claim 1, wherein the pharmacological chaperone isAc-Thr-Glu-Val-Ala-NH2.
 5. The method of claim 1, wherein the individualhas or is at risk of having one or more disease conditions associatedwith α-1-antitrypsin selected from cirrhosis, chronic obstructivepulmonary disease, pneumothorax, asthma, Wegener's granulomatosis,pancreatitis, gallstones, bronchiectasis, pelvic organ prolapse, primarysclerosing cholangitis, autoimmune hepatitis, emphysema and cancer. 6.The method of claim 1, wherein the individual has or is at risk ofhaving a liver disorder associated with alpha-1-antitrypsin.
 7. Themethod of claim 1, wherein the individual has or is at risk of having alung disorder associated with alpha-1-antitrypsin.
 8. The method ofclaim 1, wherein the individual has an E342 missense mutation inα-1-antitrypsin in at least one allele.
 9. A method for determiningmodulation of alpha-1-antitrypsin activity by a test compoundcomprising: (a) providing a labeled elastin wherein cleavage of thelabeled elastin by elastase results in a detectable signal; (b)combining the test compound, the labeled elastin, elastase andalpha-1-antitrypsin; and (c) measuring the detectable signal wherein adecrease in the detectable signal relative to that without the testcompound indicates the test compound inhibits alpha-1-antitrypsinactivity and an increase in the detectable signal relative to thatwithout the test compound indicates the test compound enhancesalpha-1-antitrypsin activity.
 10. A compound according to the structure:

wherein: R1 is —(CH₂)₂—COOH or —CH₂—OH R2 is —CH₃ or —CH—(CH₃)₂.
 11. Thecompound of claim 10, wherein R1 is —CH₂—OH and R2 is —CH₃.
 12. Thecompound of claim 10, wherein R1 is —(CH₂)₂—COOH and R2 is —CH—(CH₃)₂.13. The compound of claim 10, wherein R1 is —(CH₂)₂—COOH and R2 is —CH₃.14. A compound according to the structure:

wherein R₃ is —H or —CH₃ R₄ is —H or —OCH₃ R₅ is —H or —OH.
 15. Thecompound of claim 14 having the structure:


16. The compound of claim 14 having the structure:


17. The compound of claim 14 having the structure:


18. The compound of claim 14 having the structure:


19. The compound of claim 14 having the structure:


20. The compound of claim 14 having the structure: